The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.     

Deterioration of diacylglycerol‐ and triacylglycerol‐rich oils during frying of potatoes

The stabilities of a commercial diacylglycerol‐rich oil (DAG) and a salad oil (TAG) that had been prepared from a mixture of rapeseed and soybean oils were compared while frying potatoes at 180 °C for 3 h. The representative chemical and physical characteristics of the oils were assessed before and after frying, together with the amount of volatile aldehydes in the exhaust of frying. Among the deterioration indications, the carbonyl value, polymer content, and residual polyunsaturated fatty acid content were similar and not significantly different between the TAG and DAG. On the other hand, the characteristics relating to free fatty acids, i.e. the acid value and emission of chemiluminescence at 100 °C, were greater and the smoke and flash points were lower in the DAG than in the TAG. An irritating odor was generated from the DAG after 1 h of frying and got stronger as frying continued. These results suggested that DAG more easily forms free fatty acids under frying conditions than TAG.     

Water-retaining ability of diacylglycerol

The water-in-oil emulsification characteristics and the adsorption properties of DAG at the oil/water interface were investigated for DAG having different FA compositions. The water-retaining ability of DAG is dependent on the FA composition but is not dependent on the interfacial tension at the oil/water interface in a simple way. The water-retaining ability is very different between uni-chain DAG (two FA have the same chain length) and complex-chain DAG (one FA is oleic acid and the other has a shorter alkyl chain). Uni-chain DAG, having long FA chains (R=C₁₂ or C_18∶1) have the ability to emulsify water at the volume fraction of 80% (ϕ80%), but uni-chain DAG with short or medium chain-length FA (R=C₃, C₄, C₆, C₈) show little ability to retain water. For complex-chain DAG, all the DAG studied here (R₁=C_18∶1, R₂=C₂−C₁₂) have the ability to emulsify water at ϕ80%. The stability of the emulsions, however, varies with the chain length of the R₂ FA (R₂ stability order: C₂, C₃>C_18∶1, C₁₀>C₈>C₄, C₆). The relationship between the water-retaining ability and the molecular structure of DAG is discussed from the viewpoint of intra- and intermolecular interactions between the FA chains.     

Kinetic study of liquid lipase-catalyzed glycerolysis of olive oil using Lipozyme TL 100L

Monoacylglycerol (MAG) and diacylglycerol (DAG) are two natural components found in most edible oils and fats. Conventional synthesis of MAG and DAG is usually conducted by glycerolysis of triacylglycerol (TAG) at high temperatures (above 200°C) in the presence of an alkaline catalyst. In this work, the synthesis of MAG and DAG using enzymatic glycerolysis of olive oil was investigated using Tween 80 as surfactant, n-butanol as co-surfactant and the novel lipase in free/liquid formulation Lipozyme TL 100L as catalyst. Experimental design was used to evaluate the effect of enzyme load and reaction temperature on the feedstock conversion. Enzyme load and system temperature were significant variables in the statistical design and the best condition was found at 35°C, 7.5 vol% of Lipozyme TL 100L and glycerol to oil volumetric ratio of 2:1 with conversion of TAG at approximately 98% after 2 h of process. A mathematical model based on the Ping-Pong Bi-Bi mechanism was used to describe the reaction kinetics. The model adequately described the behavior of the system and can be a useful tool for the design of reactors in larger scales.     

Production of microparticles from milk fat products using the Supercritical Melt Micronization (ScMM) process

The ScMM (Supercritical Melt Micronization) process was applied for the production of microparticles from anhydrous milk fat (AMF) and a diacylglycerol-based modified milk fat (D-AMF). Both fats were able to dissolve ca. 30 wt% CO₂ in the studied pressure and temperature ranges, being the CO₂ amount slightly higher for AMF. A melting point depression was observed in both systems in the presence of CO₂. Two powder morphologies were obtained (spherical hollow particles and a mass sponge-like broken particles) depending on the ScMM process conditions. The concentration of CO₂ in the fat melt was the main process variable affecting the particle morphology, followed by the temperature of the melt. The small broken particles originated from the breakage of spherical fat particles that solidified before all CO₂ could escape from the atomized droplets. While the hollow spheres had a tendency to agglomerate, the broken microparticles constituted a free-flowing powder as long as they were stored at low temperatures (up to −18 °C). Both types of particles have a potential for being incorporated in refrigerated or frozen food products as a structuring agent.     

Residue Asn277 Affects the Stability and Substrate Specificity of the SMG1 Lipase from Malassezia globosa

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3₁₀4 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.     

Membrane‐Surface Anchoring of Charged Diacylglycerol‐Lactones Correlates with Biological Activities

Synthetic diacylglycerol‐lactones (DAG‐lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other nonkinase proteins that share similar C1 membrane‐targeting domains with PKC. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG‐lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α‐pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG‐lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N‐alkyl chain in the R² position of the DAG‐lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively charged DAG‐lactone derivatives play a central role in determining biological activity, and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.     

甘油二酯油对代谢综合征影响的研究进展北大核心CSCD

近年来,我国人群代谢综合征的发病率较高,国内外研究发现甘油二酯油可降低受试者的体质量,抑制餐后血脂及血糖的升高,或可有助于防控代谢综合征。为阐明甘油二酯油与代谢综合征的关系,综述了甘油二酯油对代谢综合征的影响,包括抑制餐后血脂升高,降脂减重,降低血糖、改善胰岛素敏感性,调控血压等,并对甘油二酯油的食用安全性进行了评价。甘油二酯油具有独特的代谢特征且安全性良好,是新兴膳食油脂。     

甘油二酯的合成、代谢及应用研究进展

甘油二酯是油脂代谢的中间产物,是一种重要的功能油脂,也是国际公认安全的食品成分。根据脂肪酸酰基与甘油骨架上羟基连接位置不同,甘油二酯分为1,3-甘油二酯、1,2-甘油二酯和2,3-甘油二酯三种异构体,其中1,2-甘油二酯和2,3-甘油二酯互为同分异构体。目前国内外多项研究表明甘油二酯特别是1,3-甘油二酯具有抑制脂肪积累和降低血脂的功能。因此,甘油二酯已被广泛应用于医药、食品、化工等工业领域中,具有良好的商业价值。本文主要介绍了甘油二酯的代谢、合成、检测、应用现状及伴随产生的食用安全性,并讨论了甘油二酯未来的研究方向,以期为甘油二酯的进一步研究提供参考。     

The nucleotide sequence of a 2·1 kb fragment from chromosome VI of Saccharomyces cerevisiae identifies a tRNAGly gene,part of a delta element and a palindromic sequence

The nucleotide sequence was determined of a 2·1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae. Analysis revealed the presence of a tRNA^GLy gene, part of a delta element and a remarkable palindromic sequence. The longest open reading frame found encodes a putative protein of 195 amino acids. Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase.